Microfluidic devices for electrophoretic analysis of materials

ABSTRACT

The present invention provides a microfluidic system for electrophoretic analysis of materials in the fields of chemistry, biochemistry, biotechnology, molecular biology and numerous other fields. Light absorbance signals are received by a photodetector from periodically spaced regions along a channel in the microfluidic system. The signals received by the photodetector are modulated by the movement of species bands through the channel under electrophoretic forces. By Fourier analysis, the velocity of each species band is determined, and identification of the species is made based on its electrophoretic mobility in the channel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/606,158 filed Jun. 25, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/977,056 filed Oct. 12, 2001, which is acontinuation of U.S. patent application Ser. No. 09/378,169 filed Aug.19, 1999 (now U.S. Pat. No. 6,337,740), which is a continuation of U.S.patent application Ser. No. 09/207,864 filed Dec. 8, 1998 (now U.S. Pat.No. 6,233,048), which is a continuation of U.S. patent application Ser.No. 08/941,679 filed Sep. 30, 1997 (now U.S. Pat. No. 5,852,495), whichis a continuation of U.S. patent application Ser. No. 08/683,080 filedJul. 16, 1996 (now U.S. Pat. No. 5,699,157).

BACKGROUND OF THE INVENTION

There has been a growing interest in the manufacture and use ofmicrofluidic systems for the acquisition of chemical and biochemicalinformation. Techniques commonly associated with the semiconductorelectronics industry, such as photolithography, wet chemical etching,etc., are used in the fabrication of these microfluidic systems. Theterm, “microfluidic”, refers to system or devices having channels andchambers are generally fabricated at the micron or submicron scale,e.g., having at least one cross-sectional dimension in the range of fromabout 0.1 μm to about 500 μm. Early discussions of the use of planarchip technology for the fabrication of microfluidic systems are providedin Manz et al., Trends in Anal. Chem. (1990) 10(5):144-149 and Manz etal., Avd. in Chromatog. (1993) 33:1-66, which describe the fabricationof such fluidic devices and particularly microcapillary devices, insilicon and glass substrates.

Application of microfluidic systems are myriad. For example,International Patent Appln. WO 96/04547, published Feb. 15, 1996,describes the use of microfluidic systems for capillary electrophoresis,liquid chromatography, flow injection analysis, and chemical reactionand synthesis. U.S. Pat. No. 5,942,443 entitled “HIGH THROUGHPUTSCREENING ASSAY SYSTEMS IN MICROSCALE FLUIDIC DEVICES”, filed Jun. 28,1996 by J. Wallace Parce et al. and assigned to the present assignee,discloses wide ranging applications of microfluidic systems in rapidlyassaying compounds for their effects on chemical, and preferably,biochemical systems. The phase, “biochemical system,” generally refersto a chemical interaction which involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signalling and other reactions.Biochemical systems of particular interest include, e.g.,receptor-ligand interactions, enzyme-substrate interactions, cellularsignalling pathways, transport reactions involving model barrier systems(e.g., cells or membrane fractions) for bioavailability screening, and avariety of other general systems.

As disclosed in International Patent Appln. WO 96/04547 and U.S. Pat.No. 5,942,443 noted above, one of the operations which is suitable formicrofluidic systems is capillary electrophoresis. In capillaryelectrophoresis charged molecular species, such as nucleic acids orproteins, for example, are separated in solution by an electric field.With very small capillary tubes as separation channels in a microfluidicsystem, resolution is enhanced because band broadening due to thermalconvection is minimized. The requirement of only a small amount ofsample material containing the molecular species is a further advantageof capillary electrophoresis in microfluidic systems.

Nonetheless, there is still room for improvement in capillaryelectrophoresis. One of the goals of microfluidic systems is highthroughput. Presently capillary electrophoresis in microfluidic systemsis performed by the observation of separating bands of species migratingin a separation channel under an electric field. The electrophoreticmobility of a species is determined by the time required from the entryof a test compound material into the separation channel for a speciesband from the test compound material to pass a detection point along theseparation channel. The operation is completed after the last speciesband clears the detection point. See, for example, the above-citedInternational Patent Appln. WO 96/04547. While these operations are fastcompared to macroscale electrophoretic methods, the operations fallshort of a highly automated microfluidic system, such as disclosed inthe above-mentioned Pat. No. 5,942,443, for example.

In contrast, the present invention solves or substantially mitigatesthese problems. With the present invention, the electrophoretic mobilityof each species is determined as the various species undergoelectrophoresis in a microfluidic system. Identification of each speciescan be made automatically.

SUMMARY OF THE INVENTION

The present invention provides for a microfluidic system for high-speedelectrophoretic analysis of subject materials for applications in thefields of chemistry, biochemistry, biotechnology, molecular biology andnumerous other areas. The system has a channel in a substrate, a lightsource and a photoreceptor. The channel holds subject materials insolution in an electric field so that the materials move through thechannel and separate into bands according to species. The light sourceexcites fluorescent light in the species bands and the photoreceptor isarranged to receive the fluorescent light from the bands. The systemfurther has a means for masking the channel so that the photoreceptorcan receive the fluorescent light only at periodically spaced regionsalong the channel. The system also has an unit connected to analyze themodulation frequencies of light intensity received by the photoreceptorso that velocities of the bands along the channel are determined. Thisallows the materials to be analyzed.

In accordance with the present invention, the microfluidic system canalso be arranged to operate with species bands which absorb the lightfrom the light source. The absorbance of light by the species bandscreates the modulation in light intensity which allow the velocities ofthe bands along the channel to be determined and the subject material tobe analyzed.

The present invention also provides for a method of performinghigh-speed electrophoretic analysis of subject materials. The methodcomprises the steps of holding the subject materials in solution in achannel of a microfluidic system; subjecting the materials to anelectric field so that the subject materials move through the channeland separate into species bands; directing light toward the channel;receiving light from periodically spaced regions along the channelsimultaneously; and analyzing the frequencies of light intensity of thereceived light so that velocities of the bands along the channel can bedetermined for analysis of said materials. The determination of thevelocity of a species band determines the electrophoretic mobility ofthe species and its identification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of one embodiment of a microfluidicsystem;

FIG. 2A is a representation of the details of a portion of themicrofluidic system according to one embodiment of the presentinvention; FIG. 2B is a detailed representation of a portion of theseparation channel of microfluidic system of FIG. 2A;

FIG. 3A represents an alternative arrangement of the portion of themicrofluidic system according to another embodiment of the presentinvention; FIG. 3B is a detailed representation of a portion of theseparation channel of microfluidic system of FIG. 3A; and

FIG. 4 represents still another arrangement of portion of themicrofluidic system according to another embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

General Description of Microfluidic Systems

FIG. 1 discloses a representative diagram of an exemplary microfluidicsystem 100 according to the present invention. As shown, the overalldevice 100 is fabricated in a planar substrate 102. Suitable substratematerials are generally selected based upon their compatibility with theconditions present in the particular operation to be performed by thedevice. Such conditions can include extremes of pH, temperature, saltconcentration, and application of electrical fields. Additionally,substrate materials are also selected for their inertness to criticalcomponents of an analysis or synthesis to be carried out by the system.

Useful substrate materials include, e.g., glass, quartz and silicon, aswell as polymeric substrates, e.g., plastics. In the case of conductiveor semiconductive substrates, there should be an insulating layer on thesubstrate. This is particularly important where the device incorporateselectrical elements, e.g., electrical fluid direction systems, sensorsand the like, or uses electroosmotic forces to move materials about thesystem, as discussed below. In the case of polymeric substrates, thesubstrate materials may be rigid, semi-rigid, or non-rigid, opaque,semi-opaque or transparent, depending upon the use for which they areintended. For example, devices which include an optical or visualdetection element, are generally fabricated, at least in part, fromtransparent materials to allow, or at least, facilitate that detection.Alternatively, transparent windows of, e.g., glass or quartz, may beincorporated into the device for these types detection elements.Additionally, the polymeric materials may have linear or branchedbackbones, and may be crosslinked or non-crosslinked. Examples ofparticularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate and the like.

The system shown in FIG. 1 includes a series of channels 110, 112, 114and 116 fabricated into the surface of the substrate 102. As discussedin the definition of “microfluidic,” these channels typically have verysmall cross sectional dimensions, preferably in the range from about 0.1μm to about 100 μm. For the particular applications discussed below,channels with depths of about 10 μm and widths of about 60 μm workeffectively, though deviations from these dimensions are also possible.

Manufacturing of these channels and other microscale elements into thesurface of the substrate 102 may be carried out by any number ofmicrofabrication techniques that are well known in the art. For example,lithographic techniques may be employed in fabricating glass, quartz orsilicon substrates, for example, with methods well known in thesemiconductor manufacturing industries. Photolithographic masking,plasma or wet etching and other semiconductor processing technologiesdefine microscale elements in and on substrate surfaces. Alternatively,micromachining methods, such as laser drilling, micromilling and thelike, may be employed. Similarly, for polymeric substrates, well knownmanufacturing techniques may also be used. These techniques includeinjection molding techniques or stamp molding methods where largenumbers of substrates may be produced using, e.g., rolling stamps toproduce large sheets of microscale substrates, or polymer microcastingtechniques where the substrate is polymerized within a microfabricatedmold.

Besides the substrate 102, the microfluidic system includes anadditional planar element (not shown) which overlays the channeledsubstrate 102 to enclose and fluidly seal the various channels to formconduits. The planar cover element may be attached to the substrate by avariety of means, including, e.g., thermal bonding, adhesives or, in thecase of glass, or semi-rigid and non-rigid polymeric substrates, anatural adhesion between the two components. The planar cover elementmay additionally be provided with access ports and/or reservoirs forintroducing the various fluid elements needed for a particular screen.

The system 100 shown in FIG. 1 also includes reservoirs 104, 106 and108, which are disposed and fluidly connected at the ends of thechannels 114, 116 and 110 respectively. As shown, sample channel 112, isused to introduce a plurality of different subject materials into thedevice. It should be noted that the term, “subject materials,” simplyrefers to the material, such as a chemical or biological compound, ofinterest. Subject compounds may include a wide variety of differentcompounds, including chemical compounds, mixtures of chemical compounds,e.g., polysaccharides, small organic or inorganic molecules, biologicalmacromolecules, e.g., peptides, proteins, nucleic acids, or extractsmade from biological materials, such as bacteria, plants, fungi, oranimal cells or tissues, naturally occurring or synthetic compositions.

Many methods have been described for the transport and direction offluids, e.g., samples, analytes, buffers and reagents, withinmicrofluidic systems or devices. One method moves fluids withinmicrofabricated devices by mechanical micropumps and valves within thedevice. See, published U.K. Patent Application No. 2 248 891 (Oct. 18,1990), published European Patent Application No. 568 902 (May 2, 1992),U.S. Pat. Nos. 5,271,724 (Aug. 21, 1991) and 5,277,556 (Jul. 3, 1991).See also, U.S. Pat. No. 5,171,132 (Dec. 21, 1990) to Miyazaki et al.Another method uses acoustic energy to move fluid samples within devicesby the effects of acoustic streaming. See, published PCT Application No.94/05414 to Northrup and White. A straightforward method appliesexternal pressure to move fluids within the device. See, e.g., thediscussion in U.S. Pat. No. 5,304,487 to Wilding et al.

While these methods could be used to transfer the test compoundmaterials to the separation channel for electrophoresis, a preferablemethod uses electric fields to move fluid materials through the channelsof a microfluidic system. See, e.g., published European PatentApplication No. 376 611 (Dec. 30, 1988) to Kovacs, Harrison et al.,Anal. Chem. (1992) 64:1926-1932 and Manz et al. J. Chromatog. (1992)593:253-258, U.S. Pat. No. 5,126,022 to Soane. Electrokinetic forceshave the advantages of direct control, fast response and simplicity.Furthermore, the use of electrokinetic forces to move the subjectmaterials about the channels of the microfluidic system 100 isconsistent with the use of electrophoretic forces in the separationchannel 110.

To provide such electrokinetic transport, the system 100 includes avoltage controller that is capable of applying selectable voltagelevels, simultaneously, to each of the reservoirs, including ground.Such a voltage controller can be implemented using multiple voltagedividers and multiple relays to obtain the selectable voltage levels.Alternatively, multiple independent voltage sources may be used. Thevoltage controller is electrically connected to each of the reservoirsvia an electrode positioned or fabricated within each of the pluralityof reservoirs. See, for example, published International PatentApplication No. WO 96/04547 to Ramsey, which is incorporated herein byreference in its entirety for all purposes.

Alternatively, rather than voltage, another electrical parameter, suchas current, may be used to control the flow of fluids through thechannels. A description of such alternate electrical parametric controlis found in U.S. Pat. No. 5,800,690, entitled “VARIABLE CONTROL OFELECTROOSMOTIC AND/OR ELECTROPHORETIC FORCES WITHIN A FLUID-CONTAININGSTRUCTURE VIA ELECTRICAL FORCES”, filed Jul. 3, 1996 by Calvin Y. H.Chow and J. Wallace Parce and assigned to the present assignee. Thisapplication is incorporated herein by reference in its entirety for allpurposes.

Stated more precisely, electrokinetic forces may be separated intoelectroosmotic forces and electrophoretic forces. The fluid controlsystems used in the system of the present invention employelectroosmotic force to move, direct and mix fluids in the variouschannels and reaction chambers present on the surface of the substrate102. In brief, when an appropriate fluid is placed in a channel or otherfluid conduit having functional groups present at the surface, thosegroups can ionize. For example, where the surface of the channelincludes hydroxyl functional groups at the surface, protons can leavethe surface of the channel and enter the fluid. Under such conditions,the surface possesses a net negative charge, whereas the fluid possessesan excess of protons or positive charge, particularly localized near theinterface between the channel surface and the fluid.

By applying an electric field across the length of the channel, cationsflow toward the negative electrode. Movement of the positively chargedspecies in the fluid pulls the solvent with them. The steady statevelocity of this fluid movement is generally given by the equation:$v = \frac{ɛ\quad\xi\quad E}{4\quad\pi\quad\eta}$where v is the solvent velocity, ε is the dielectric constant of thefluid, ξ is the zeta potential of the surface, E is the electric fieldstrength, and η is the solvent viscosity. Thus, as can be easily seenfrom this equation, the solvent velocity is directly proportional to thezeta potential and the applied field.

Besides electroosmotic forces, there are also electrophoretic forceswhich affect charged molecules as they move through the system 100. Inthe transport of subject materials from one point to another point inthe system 100, it is often desirable for the composition of the subjectmaterials to remain unaffected in the transport, i.e., that the subjectmaterials are not electrophoretically differentiated in the transportuntil desired. To do so, the subject materials are transported in fluidslug regions 120 of predetermined ionic concentrations. The regions areseparated by buffer regions of varying ionic concentrations andrepresented by buffer regions 121 in FIG. 1. A related patentapplication, U.S. Pat. No. 5,779,868, entitled “ELECTROPIPETTOR ANDCOMPENSATION MEANS FOR ELECTROPHORETIC BIAS,” filed Jun. 28, 1996 by J.Wallace Parce and Michael R. Knapp, and assigned to the presentassignee, explains various arrangements of slugs, and buffer regions ofhigh and low ionic concentrations in transporting subject materials withelectrokinetic forces. The application is incorporated herein byreference in its entirety for all purposes. The application alsoexplains how the channel 112 may be fluidly connected to a source oflarge numbers of separate subject materials which are individuallyintroduced into the sample channel 112 and subsequently into theseparation channel 110 for analysis.

Electrophoresis in Microfluidic System and Operation

As described in the above-cited International Patent Appln. WO 96/04547and the previously mentioned U.S. Pat. No. 5,942,443 entitled “HIGHTHROUGHPUT SCREENING ASSAY SYSTEMS IN MICROSCALE FLUIDIC DEVICES”, thedisclosures of which are incorporated herein by reference for allpurposes, the slugs 120 of subject materials, separated by buffers 121,are moved through the sample channel 112 and into the separation channel110. Each slug 120 is subjected to an electric field in the channel 110so that the constituent species in each slug 120 separates into speciesbands 123, as shown in FIG. 1.

When the slugs 120 of subject materials are placed in the separationchannel 110, the materials are subjected to an electric field bycreating a large potential difference between the terminals in thereservoir 104 and 108. The species in the slugs separate according totheir electric charges and sizes of their molecules. The species aresubjected to electric fields in the range of 200 volts/cm. In accordancewith one aspect of the present invention, the species are labeled withfluorescent label materials, such as fluorescent intercalating agents,such as ethidium bromide for polynucleotides, or fluoresceinisothiocyanate or fluorescamine for proteins, as is typically done withconventional electrophoresis.

As shown in FIG. 2A, the arrangement has a light source 120, a firstlens 124, a mask 122, the separation channel 10, a second lens 126, afilter 128, and a photoreceptor 130 connected to a frequency analyzerunit 134. The light source 120 emits light at wavelengths to energizethe fluorescent labels of the species in the separation channel 110.Lamps, lasers and light-emitting diodes may be used for the source 120.The mask 122 is located between the light source 120 and the separationchannel 110 and blocks light from reaching selected portions of thechannel 110.

The projection of the mask 122 by the light source 120 onto theseparation channel 110 results in a series of alternating illuminatedand darkened regions which are equally spaced along the channel 110.Each darkened region 140 has the same width as another darkened regionalong the separation channel 110 and is approximately the same width asthe species bands 123 in the separation channel 110, as shown in FIG.2B. The illuminated regions 142 along the separation channel 110 arealso approximately the same width as the darkened regions 140. Forexample, with a separation column approximately 10 μm deep and 60 μmwide, the illuminated and darkened regions 142 and 140 are approximately50-500 μm along the separation channel 110.

As each species band from the sample slugs travel through thealternating darkened and illuminated regions 140 and 142 respectively,the species bands 123 are alternately fluorescent in the illuminatedregions 142 and unlit in the darkened regions 140. As each speciestravels down the separating channel 110, the species fluoresces off andon with a characteristic frequency corresponding to its velocity alongthe channel 110. The velocity, v, of the particular species is directlyrelated to the electrophoretic mobility, μ_(ep), of that species:v=μ _(ep) ·Ewhere E is the electric field. Thus a plurality of different speciesmoving through the separation channel 110 fluoresces at a plurality offrequencies, each corresponding to a particular species.

The light from the separation channel 110 is focussed by the lens 126upon the photoreceptor 130. The light received by the photoreceptor 130,which may be a photomultiplier tube, a photodiode, a CCD array, and thelike, is converted into electrical signals which are, in turn, sent tothe frequency analyzer unit 134. The frequency analyzer unit 134, bystraightforward Fourier analysis, breaks the electrical signals intotheir component frequencies. These electrical signal frequencies are thesame as that of the modulated light intensities generated by the speciesundergoing electrophoresis in the separation channel 110. The frequencyof light intensity is related to the electrophoretic mobility of eachspecies band. Hence, a computer unit with a calibrated look-up table canautomatically identify each species according to its electrical signalfrequency from the frequency analyzer unit 134. The electrophoresisoperation is entirely automated.

Note that each species band 123 need not pass completely through theseparation channel 110. Identification occurs as soon as acharacteristic optical modulation frequency is generated after thespecies passes through a predetermined number of alternating darkenedand illuminated regions in the channel 110. Thus electrophoresis isperformed in a matter of seconds.

As stated above, the mask 122 is arranged such that the alternatingdarkened and illuminated regions are approximately the same width alongthe separation channel 110 with respect to each other and to the widestspecies band. This ensures the largest possible variation between themaxima and minima of light intensity from the fluorescent species bandspassing through the mask regions.

As symbolically shown in FIG. 2A, the photoreceptor 130 is placed alongan axis formed with the light source 120, the mask 122 and the lens 126.An alternative arrangement has the light source 120 and the mask 122 offthe axis so that light from the source 120 directed toward theseparation channel 110 is also directed away from the photoreceptor 130.This arrangement allows the photoreceptor 130 to be illuminated only bythe fluorescent light from the labeled species in the channel 110.Furthermore, to avoid contamination of the optical signals received bythe photoreceptor 130, a filter 128 may be used for the photoreceptor130. The filter 128 is a band-pass filter transmitting light only atwavelengths emitted by the fluorescent species, and blocking light atother wavelengths, i.e., light from the source 120. Alternatively, thefilter 128 might be selective toward blocking light at the light sourcewavelengths. Typically, the fluorescent label materials fluoresce atlonger wavelengths than those of the source 120. For example, forpolynucleotides labeled with ethidium bromide as subject materials forelectrophoresis, a light source emitting light at 540 nm is used and thespecies bands fluoresce at 610 nm. For proteins labelled withfluorescein, a light source at 490 nm works with species bandsfluorescing at 525 nm.

As described above, the mask 122 is projected onto the separationchannel 110. An alternative arrangement imposes the mask 122 onto thesubstrate itself so that a series of alternating darkened and lightregions are created along the channel 110. Such an arrangement isillustrated in FIG. 3A. The light source 120 illuminates the speciesbands 122 in the separation channel 110 directly. On the side of thechannel 110 toward the photoreceptor 120, a mask 150 of alternatingdarkened and transparent regions 154 and 152 respectively are placed onthe substrate 152, as shown in FIG. 3B. The dimensions and spacing ofthe regions 154 and 152 are the same as the projection of the mask 122in FIGS. 2A and 2B.

Still another arrangement projects the fluorescent species bands 123 inthe separation channel 110 unto a mask 160, as shown in FIG. 4. Afterbeing collimated by a lens 164, light from the source 120 illuminatesthe species bands 123. Since light fluoresces from the bands 123isotropically, the light is projected toward the mask 160 through afocussing lens 165. Light from the other side of the mask 160 is focusedby the lens 126 onto the photodetector 120. As explained above, theelements of FIG. 4 illustrate a general relationship with each other.The lens 165, mask 160, lens 126, filter 128, and photoreceptor 130 neednot be aligned with source 120, lens 164 and channel 110.

The arrangements above analyze the subject materials undergoingelectrophoresis by the reception of fluorescent light from the movingspecies bands 123. The present invention also operates with theabsorbance of light by the subject material. For example, using thearrangement of FIG. 2A, the light source 120 is selected to radiatelight at wavelengths which are absorbed by the subject material. Forproteins, the light source 120 may operate at wavelengths of 280 nm, forexample. For polynucleotides, 260 nm is a suitable wavelength for thelight source 120. The lens 126, filter 128 and photoreceptor arearranged to receive the light from the source 120 through the mask 122and channel 120. The light source 120, lens 124, mask 122, channel 110,lens 126, filter 128 and photoreceptor 130 are optically aligned and thefilter 128 is selected to pass light of the wavelength of interest fromthe source 120 to the photoreceptor 130. More typically for absorptionmeasurements, the filter 128 is placed next to the source.

Rather than light from the species bands 123, darkness from thelight-absorbing bands 123 moving in the channel 110 causes a varyingsignal to be received by the photoreceptor 130. Fourier analysis of thesignal ultimately identifies the species in the channel 110. Similarly,the embodiments of the present invention illustrated in FIGS. 3A and 4can be adapted to light absorbance by the species bands 123, rather thanlight fluorescence.

In another embodiment of the present invention, the mask 122 iseliminated. For example, a coherent light source, such as a laser, isused for the source 120 and a pair of slits are located between thesource 120 and the channel 110. The slits are parallel to each other andperpendicular to the length of channel 110. By interference between thelight emanating from the two slits, the light falls in intensities ofalternating minima and maxima along the channel 110, like the operationof the mask 122 described previously. Light received from theperiodically spaced locations of maxima allow the determination of thevelocities of moving species bands 123 by the frequency analysis of thelight intensity modulating in time, as described previously. Thisarrangement operates in either fluorescing or absorbing mode. Of course,other arrangements with one or more light sources 120 may also createlight patterns of minima and maxima intensities along the channel 110without a mask.

Speed and sensitivity of the present invention are much enhanced overprevious systems which perform electrophoresis by the measurement of aspecies band past a detection point. The present invention has a highersignal-to-noise ratio since the light signals from the fluorescent bands123 are averaged over time by the movement of the light signals past themask regions, in contrast to a single observation at the detectionpoint.

Of course, the present invention also has the other advantages ofmicrofluidic systems, such as speed, low cost due to the low consumptionof materials and the low use of skilled labor, and accuracy. Themicrofluidic system 100 has little or no contamination with highreproducibility of results.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1. A method of detecting species bands in a separation conduit,comprising: directing light from a light source through a mask at aplurality of discrete regions of the separation conduit, the pluralityof discrete regions containing a plurality of species bands, the lightsource selected to radiate at a wavelength that is absorbed by thespecies bands; and simultaneously detecting light absorbance from saidplurality of species bands in said plurality of discrete regions.
 2. Themethod of claim 1, wherein the plurality of species bands areelectrophoresed through the separation conduit during said directing andsimultaneously detecting steps.
 3. The method of claim 2, wherein lightabsorbance from the plurality of species bands moving in the separationconduit causes a varying signal to be detected.
 4. The method of claim3, further comprising determining a rate at which the plurality ofspecies bands are moving in the separation conduit by monitoring afrequency at which light absorbance is detected in the plurality ofregions of the separation conduit.
 5. The method of claim 1, wherein theseparation conduit comprises a channel in a microfluidic device.
 6. Themethod of claim 1, wherein the species bands comprise nucleic acids. 7.The method of claim 6, wherein the light source is selected to radiateat a wavelength of 280 nm.
 8. The method of claim 1, wherein the speciesbands comprise polynucleotides.
 9. The method of claim 8, wherein thelight source is selected to radiate at a wavelength of 260 nm.
 10. Amethod of detecting species bands in a separation conduit, comprising:directing light from a light source at a plurality of discrete regionsof the separation conduit, the plurality of discrete regions containinga plurality of species bands, the light source selected to radiate at awavelength that is absorbed by the species bands; and simultaneouslydetecting light absorbance from said plurality of species bands in saidplurality of discrete regions.
 11. The method of claim 10, wherein theplurality of discrete regions are each between about 50 μm and 500 μmlong along the separation conduit.
 12. The method of claim 10, whereinthe light is direct through a pair of slits.
 13. The method of claim 12,wherein the slits are parallel to each other and perpendicular to thelength of the separation conduit.
 14. The method of claim 12, whereinthe light source is a coherent light source.
 15. The method of claim 10,wherein the separation conduit comprises a channel in a microfluidicdevice.
 16. A system for detecting separated species bands, comprising:a separation conduit for separating species bands; a light absorbancedetection system, comprising: a light source for directing light at aplurality of regions of the separation conduit at a wavelength that isabsorbed by the species bands; a mask positioned between the lightsource and the separation conduit for directing the light at a pluralityof discrete regions of the separation conduit; and a detector orientedto simultaneously detect light absorbance from the plurality of regionsof the separation conduit.
 17. The system of claim 16, wherein theseparation conduit comprises a channel in a microfluidic device.
 18. Thesystem of claim 16, wherein the detector is selected from a groupconsisting of a photomultiplier tube, a photodiode, and a CCD array. 19.The system of claim 16, further comprising: a computer unit fordetermining a rate at which the plurality of species bands are moving inthe separation conduit by monitoring a frequency at which lightabsorbance is detected in the plurality of regions of the separationconduit.
 20. The system of claim 16, further comprising: a computer unitwith a calibrated look-up table.